Non-peptide small-molecule inhibitors designed to target the MDM2-p53 interaction may have a great therapeutic potential for the treatment of many types of human cancer with wild-type p53. Using a powerful structure-based strategy, we have now designed a class of highly potent, non-peptide, drug-like, small- molecule inhibitors of the MDM2-p53 interaction. Based upon our promising preliminary results, we propose to further optimize this class of inhibitors, to evaluate their therapeutic potential in vitro and in vivo, and to elucidate their mechanisms of action in vitro and and in vivo. Our long-term goal in this project is to develop potent and specific non-peptide small-molecule inhibitors of the MDM2-p53 interaction for the treatment of human cancer. To achieve our goal, we will pursue the following Specific Aims: Aim 1: (a).Performance of computational structure-based design of new inhibitors to further optimize this class of promising compounds; (b).Determination of high resolution three-dimensional structures of potent inhibitors complexed with MDM2 protein through x-ray crystallography. Aim 2: Chemical synthesis of inhibitors designed in Aim 1 and biotin-labeled compounds. Aim 3: Determination of their binding and specificity to MDM2 protein using complementary fluorescence- polarization-based and NMR-based methods. Aim 4: Evaluation of their cellular activity and specificity in human cancer cells with wild-type, or mutated or deleted p53 status and selectivity over normal cells, and elucidation of their cellular mechanisms of action. Aim 5: Determination of the therapeutic potential of several of the most promising MDM2 inhibitors in animal models of human cancer and elucidation of their in vivo mechanisms of action. With these tasks successfully completed, we will be able to bring a class of highly potent and optimizednon- peptide small-molecule inhibitors of the MDM2-p53 interaction into advanced preclinical development. It is predicted that such potent and specific small-molecule inhibitor of the MDM2-p53 interaction will be effective for the treatment of many types of human cancer with wild-type p53 status.